Immune Activation following Irbesartan Treatment in a Colorectal Cancer Patient: A Case Study.

Colorectal cancers are one of the most prevalent tumour types worldwide and, despite the emergence of targeted and biologic therapies, have among the highest mortality rates. The Personalized OncoGenomics (POG) program at BC Cancer performs whole genome and transcriptome analysis (WGTA) to identify specific alterations in an individual's cancer that may be most effectively targeted. Informed using WGTA, a patient with advanced mismatch repair-deficient colorectal cancer was treated with the antihypertensive drug irbesartan and experienced a profound and durable response. We describe the subsequent relapse of this patient and potential mechanisms of response using WGTA and multiplex immunohistochemistry (m-IHC) profiling of biopsies before and after treatment from the same metastatic site of the L3 spine. We did not observe marked differences in the genomic landscape before and after treatment. Analyses revealed an increase in immune signalling and infiltrating immune cells, particularly CD8+ T cells, in the relapsed tumour. These results indicate that the observed anti-tumour response to irbesartan may have been due to an activated immune response. Determining whether there may be other cancer contexts in which irbesartan may be similarly valuable will require additional studies.

Whole-genome and transcriptome analysis enhances precision cancer treatment options.

BACKGROUND: Recent advances are enabling delivery of precision genomic medicine to cancer clinics. While the majority of approaches profile panels of selected genes or hotspot regions, comprehensive data provided by whole-genome and transcriptome sequencing and analysis (WGTA) present an opportunity to align a much larger proportion of patients to therapies. PATIENTS AND METHODS: Samples from 570 patients with advanced or metastatic cancer of diverse types enrolled in the Personalized OncoGenomics (POG) program underwent WGTA. DNA-based data, including mutations, copy number and mutation signatures, were combined with RNA-based data, including gene expression and fusions, to generate comprehensive WGTA profiles. A multidisciplinary molecular tumour board used WGTA profiles to identify and prioritize clinically actionable alterations and inform therapy. Patient responses to WGTA-informed therapies were collected. RESULTS: Clinically actionable targets were identified for 83% of patients, of which 37% of patients received WGTA-informed treatments. RNA expression data were particularly informative, contributing to 67% of WGTA-informed treatments; 25% of treatments were informed by RNA expression alone. Of a total 248 WGTA-informed treatments, 46% resulted in clinical benefit. RNA expression data were comparable to DNA-based mutation and copy number data in aligning to clinically beneficial treatments. Genome signatures also guided therapeutics including platinum, poly-ADP ribose polymerase inhibitors and immunotherapies. Patients accessed WGTA-informed treatments through clinical trials (19%), off-label use (35%) and as standard therapies (46%) including those which would not otherwise have been the next choice of therapy, demonstrating the utility of genomic information to direct use of chemotherapies as well as targeted therapies. CONCLUSIONS: Integrating RNA expression and genome data illuminated treatment options that resulted in 46% of treated patients experiencing positive clinical benefit, supporting the use of comprehensive WGTA profiling in clinical cancer care.

Successful targeting of the NRG1 pathway indicates novel treatment strategy for metastatic cancer.

BACKGROUND: NRG1 fusion-positive lung cancers have emerged as potentially actionable events in lung cancer, but clinical support is currently limited and no evidence of efficacy of this approach in cancers beyond lung has been shown. PATIENTS AND METHODS: Here, we describe two patients with advanced cancers refractory to standard therapies. Patient 1 had lung adenocarcinoma and patient 2 cholangiocarcinoma. Whole-genome and transcriptome sequencing were carried out for these cases with select findings validated by fluorescence in situ hybridization. RESULTS: Both tumors were found to be positive for NRG1 gene fusions. In patient 1, an SDC4-NRG1 gene fusion was detected, similar gene fusions having been described in lung cancers previously. In patient 2, a novel ATP1B1-NRG1 gene fusion was detected. Cholangiocarcinoma is not a disease type in which NRG1 fusions had been described previously. Integrative genome analysis was used to assess the potential functional significance of the detected genomic events including the gene fusions, prioritizing therapeutic strategies targeting the HER-family of growth factor receptors. Both patients were treated with the pan HER-family kinase inhibitor afatinib and both displayed significant and durable response to treatment. Upon progression sites of disease were sequenced. The lack of obvious genomic events to describe the disease progression indicated that broad transcriptomic or epigenetic mechanisms could be attributed to the lack of prolonged response to afatinib. CONCLUSION: These observations lend further support to the use of pan HER-tyrosine kinase inhibitors for the treatment of NRG1 fusion-positive in both cancers of lung and hepatocellular origin and indicate more broadly that cancers found to be NRG1 fusion-positive may benefit from such a clinical approach regardless of their site of origin. CLINICAL TRIAL INFORMATION: Personalized Oncogenomics (POG) Program of British Columbia: Utilization of Genomic Analysis to Better Understand Tumour Heterogeneity and Evolution (NCT02155621).

Response to angiotensin blockade with irbesartan in a patient with metastatic colorectal cancer.

BACKGROUND: A patient suffering from metastatic colorectal cancer, treatment-related toxicity and resistance to standard chemotherapy and radiation was assessed as part of a personalized oncogenomics initiative to derive potential alternative therapeutic strategies. PATIENTS AND METHODS: Whole-genome and transcriptome sequencing was used to interrogate a metastatic tumor refractory to standard treatments of a patient with mismatch repair-deficient metastatic colorectal cancer. RESULTS: Integrative genomic analysis indicated overexpression of the AP-1 transcriptional complex suggesting experimental therapeutic rationales, including blockade of the renin-angiotensin system. This led to the repurposing of the angiotensin II receptor antagonist, irbesartan, as an anticancer therapy, resulting in the patient experiencing a dramatic and durable response. CONCLUSIONS: This case highlights the utility of comprehensive integrative genomic profiling and bioinformatics analysis to provide hypothetical rationales for personalized treatment options.

Personalized oncogenomics in the management of gastrointestinal carcinomas-early experiences from a pilot study.

BACKGROUND: Gastrointestinal carcinomas are genomically complex cancers that are lethal in the metastatic setting. Whole-genome and transcriptome sequencing allow for the simultaneous characterization of multiple oncogenic pathways. METHODS: We report 3 cases of metastatic gastrointestinal carcinoma in patients enrolled in the Personalized Onco-Genomics program at the BC Cancer Agency. Real-time genomic profiling was combined with clinical expertise to diagnose a carcinoma of unknown primary, to explore treatment response to bevacizumab in a colorectal cancer, and to characterize an appendiceal adenocarcinoma. RESULTS: In the first case, genomic profiling revealed an IDH1 somatic mutation, supporting the diagnosis of cholangiocarcinoma in a malignancy of unknown origin, and further guided therapy by identifying epidermal growth factor receptor amplification. In the second case, a BRAF V600E mutation and wild-type KRAS profile justified the use of targeted therapies to treat a colonic adenocarcinoma. The third case was an appendiceal adenocarcinoma defined by a p53 inactivation; Ras/raf/mek, Akt/mtor, Wnt, and notch pathway activation; and overexpression of ret, erbb2 (her2), erbb3, met, and cell cycle regulators. SUMMARY: We show that whole-genome and transcriptome sequencing can be achieved within clinically effective timelines, yielding clinically useful and actionable information.

Identification and analysis of murine pancreatic islet enhancers.

AIMS/HYPOTHESIS: The paucity of information on the epigenetic barriers that are blocking reprogramming protocols, and on what makes a beta cell unique, has hampered efforts to develop novel beta cell sources. Here, we aimed to identify enhancers in pancreatic islets, to understand their developmental ontologies, and to identify enhancers unique to islets to increase our understanding of islet-specific gene expression. METHODS: We combined H3K4me1-based nucleosome predictions with pancreatic and duodenal homeobox 1 (PDX1), neurogenic differentiation 1 (NEUROD1), v-Maf musculoaponeurotic fibrosarcoma oncogene family, protein A (MAFA) and forkhead box A2 (FOXA2) occupancy data to identify enhancers in mouse islets. RESULTS: We identified 22,223 putative enhancer loci in in vivo mouse islets. Our validation experiments suggest that nearly half of these loci are active in regulating islet gene expression, with the remaining regions probably poised for activity. We showed that these loci have at least nine developmental ontologies, and that islet enhancers predominately acquire H3K4me1 during differentiation. We next discriminated 1,799 enhancers unique to islets and showed that these islet-specific enhancers have reduced association with annotated genes, and identified a subset that are instead associated with novel islet-specific long non-coding RNAs (lncRNAs). CONCLUSIONS/INTERPRETATIONS: Our results indicate that genes with islet-specific expression and function tend to have enhancers devoid of histone methylation marks or, less often, that are bivalent or repressed, in embryonic stem cells and liver. Further, we identify a subset of enhancers unique to islets that are associated with novel islet-specific genes and lncRNAs. We anticipate that these data will facilitate the development of novel sources of functional beta cell mass.

Identification of genes targeted by the androgen and PKA signaling pathways in prostate cancer cells.

Progression of prostate cancer to androgen independence is suspected to involve the androgen and protein kinase A (PKA) signaling pathways. Here for the first time, the transcriptomes associated with each pathway and common transcriptional targets in response to stimulation of both pathways were identified in human prostate cancer cells using Affymetrix GeneChip technology (Human Genome U133 plus2). Statistically significant changes in the levels of 858 genes in response to androgen and 303 genes in response to activation of the PKA pathway were determined using GeneSpring software. Expression of a subset of these genes (22) that were transcriptional targets for the androgen and/or PKA pathways were validated by reverse transcriptase-polymerase chain reaction and Western blot analyses. Application of small interfering RNAs to the androgen receptor (AR) revealed that in addition to KLK3, levels of expression of KLK2 and SESN1 were regulated by AR activated by both the androgen and PKA signaling pathways. SESN1 was identified as a gene repressed by activated AR. These results provide a broad view of the effects of the androgen and PKA signaling pathways on the transcriptional program of prostate cancer cells and indicate that only a limited number of genes are targeted by cross-talk between AR and PKA pathways.

ORegAnno: an open access database and curation system for literature-derived promoters, transcription factor binding sites and regulatory variation.

MOTIVATION: Our understanding of gene regulation is currently limited by our ability to collectively synthesize and catalogue transcriptional regulatory elements stored in scientific literature. Over the past decade, this task has become increasingly challenging as the accrual of biologically validated regulatory sequences has accelerated. To meet this challenge, novel community-based approaches to regulatory element annotation are required. SUMMARY: Here, we present the Open Regulatory Annotation (ORegAnno) database as a dynamic collection of literature-curated regulatory regions, transcription factor binding sites and regulatory mutations (polymorphisms and haplotypes). ORegAnno has been designed to manage the submission, indexing and validation of new annotations from users worldwide. Submissions to ORegAnno are immediately cross-referenced to EnsEMBL, dbSNP, Entrez Gene, the NCBI Taxonomy database and PubMed, where appropriate. AVAILABILITY: ORegAnno is available directly through MySQL, Web services, and online at http://www.oreganno.org. All software is licensed under the Lesser GNU Public License (LGPL).

cisRED: a database system for genome-scale computational discovery of regulatory elements.

We describe cisRED, a database for conserved regulatory elements that are identified and ranked by a genome-scale computational system (www.cisred.org). The database and high-throughput predictive pipeline are designed to address diverse target genomes in the context of rapidly evolving data resources and tools. Motifs are predicted in promoter regions using multiple discovery methods applied to sequence sets that include corresponding sequence regions from vertebrates. We estimate motif significance by applying discovery and post-processing methods to randomized sequence sets that are adaptively derived from target sequence sets, retain motifs with p-values below a threshold and identify groups of similar motifs and co-occurring motif patterns. The database offers information on atomic motifs, motif groups and patterns. It is web-accessible, and can be queried directly, downloaded or installed locally.

Full-genome RNAi profiling of early embryogenesis in Caenorhabditis elegans.

A key challenge of functional genomics today is to generate well-annotated data sets that can be interpreted across different platforms and technologies. Large-scale functional genomics data often fail to connect to standard experimental approaches of gene characterization in individual laboratories. Furthermore, a lack of universal annotation standards for phenotypic data sets makes it difficult to compare different screening approaches. Here we address this problem in a screen designed to identify all genes required for the first two rounds of cell division in the Caenorhabditis elegans embryo. We used RNA-mediated interference to target 98% of all genes predicted in the C. elegans genome in combination with differential interference contrast time-lapse microscopy. Through systematic annotation of the resulting movies, we developed a phenotypic profiling system, which shows high correlation with cellular processes and biochemical pathways, thus enabling us to predict new functions for previously uncharacterized genes.

Cryptococcus neoformans gene expression during experimental cryptococcal meningitis.

Cryptococcus neoformans, an encapsulated basidiomycete fungus of medical importance, is capable of crossing the blood-brain barrier and causing meningitis in both immunocompetent and immunocompromised individuals. To gain insight into the adaptation of the fungus to the host central nervous system (CNS), serial analysis of gene expression (SAGE) was used to characterize the gene expression profile of C. neoformans cells recovered from the CNS of infected rabbits. A SAGE library was constructed, and 49,048 tags were sequenced; 16,207 of these tags were found to represent unique sequences or tag families. Of the 304 most-abundant tags, 164 were assigned to a putative gene for subsequent functional grouping. The results (as determined according to the number of tags that identified genes encoding proteins required for these functions) indicated that the C. neoformans cells were actively engaged in protein synthesis, protein degradation, stress response, small-molecule transport, and signaling. In addition, a high level of energy requirement of the fungal cells was suggested by a large number of tags that matched putative genes for energy production. Taken together, these findings provide the first insight into the transcriptional adaptation of C. neoformans to the host environment and identify the set of fungal genes most highly expressed during cerebrospinal fluid infection.

Conservation of long-range synteny and microsynteny between the genomes of two distantly related nematodes.

BACKGROUND: Comparisons between the genomes of the closely related nematodes Caenorhabditis elegans and Caenorhabditis briggsae reveal high rates of rearrangement, with a bias towards within-chromosome events. To assess whether this pattern is true of nematodes in general, we have used genome sequence to compare two nematode species that last shared a common ancestor approximately 300 million years ago: the model C. elegans and the filarial parasite Brugia malayi. RESULTS: An 83 kb region flanking the gene for Bm-mif-1 (macrophage migration inhibitory factor, a B. malayi homolog of a human cytokine) was sequenced. When compared to the complete genome of C. elegans, evidence for conservation of long-range synteny and microsynteny was found. Potential C. elegans orthologs for II of the 12 protein-coding genes predicted in the B. malayi sequence were identified. Ten of these orthologs were located on chromosome I, with eight clustered in a 2.3 Mb region. While several, relatively local, intrachromosomal rearrangements have occurred, the order, composition, and configuration of two gene clusters, each containing three genes, was conserved. Comparison of B. malayi BAC-end genome survey sequence to C. elegans also revealed a bias towards intrachromosome rearrangements. CONCLUSIONS: We suggest that intrachromosomal rearrangement is a major force driving chromosomal organization in nematodes, but is constrained by the interdigitation of functional elements of neighboring genes.

Assembly of fingerprint contigs: parallelized FPC.

SUMMARY: One of the more common uses of the program FingerPrint Contigs (FPC) is to assemble random restriction digest 'fingerprints' of overlapping genomic clones into contigs. To improve the rate of assembling contigs from large fingerprint databases we have adapted FPC so that it can be run in parallel on multiple processors and servers. The current version of 'parallelized FPC' has been used in our laboratory to assemble mammalian BAC fingerprint databases, each containing more than 300000 BAC fingerprints. AVAILABILITY: This parallelized version of FPC is available under the GNU GPL licence, and can be downloaded from ftp://ftp.bcgsc.bc.ca/pub/fpcd.